The invention relates to a process of immobilization of nucleic acid molecules on a substrate, immobilized nucleic acid obtainable therefrom and use thereof.
Immobilization (binding) of nucleic acid molecules on a substrate, such as a solid surface, is a well known problem in a large number of applications. The binding of nucleic acid molecules on substrates is of high interest for the development of nucleic acid based nanotechnology, including nucleic acid based nanoelectronics, like wires, biosensors, chips, see Storhoff, J. J., Mirkin, C. A. (1999) Chem. Rev., 99, 1849-1862 xe2x80x9cProgrammed Materials Synthesis with DNA.xe2x80x9d
Another strong motivation for immobilizing nucleic acid molecules on substrates and membranes is the characterization and engineering of nucleic acids in the field of medicine and biology, see Allison, D. P., Bottomley, L. A., Thundat, T., Brown, G. M., Woychik, R. P., Schrick, J. J., Jacobson, K. B. and Warmack, R. J. (1992) Proc. Natl. Acad. Sci. USA, 89,21 10129-10133 xe2x80x9cImmobilization of DNA for scanning probe microscopyxe2x80x9d; Bezanilla, M., Manne, S., Laney, D. E., Lyubchenko, Y. L. and Hansma, H. G. (1995) Langmuir, 11, 655-659 xe2x80x9cAdsorption of DNA to mica, silylated mica and minerals: characterization by atomic force microscopy.xe2x80x9d
Also, purification of nucleic acid solutions by attachment of nucleic acid molecules to substrates is of interest, see U.S. Pat. Nos. 5,523,392 and 5,503,816.
The binding problem for nucleic acids to solid surfaces has so far been solved by using a number of different approaches.
The most common ones employ the modification of the substrate surface by chemical treatment. One useful approach is the silanization of surfaces leading, for example, to exposed vinyl groups which bind to nucleic acid molecules, see Bensimon, D., Simon, A. J., Croquette, V., Bensimon, A. (1995) Physical Review Letters 74, 23, 4754-4757 xe2x80x9cStretching DNA with a Receding Meniscus: Experiments and Models.xe2x80x9d On mica substrates, effective binding of the nucleic acid was found by using the counterion method: this method is done by adsorbing the nucleic acid onto mica in the presence of a divalent (+2 charged) ion, like Mg2+. The idea is that the counterion will provide binding to the negatively charged nucleic acid backbone and at the same time also to the negatively charged mica surface, see Ye, J. Y., Umemura, K., Ishikawa, M., Kuroda, R. (2000) Analytical Biochemistry 281, 21-25 xe2x80x9cAtomic Force Microscopy of DNA Molecules Stretched by Spin-Coating Technique.xe2x80x9d; Dunlap, D. D., Maggi, A., Soria, M. R., Monaco, L. (1997) Nucl. Acid Res. 25, 3095 xe2x80x9cNanoscopic Structure of DNA Condensed for Gene Delivery.xe2x80x9d; Lyubchenko, Y. L., Shlyakhtenko, L. S. (1997) Proc. Natl. Acad. Sci. USA 94,496 xe2x80x9cDirect Visualization of Supercoiled DNA in situ with Atomic Force Microscopy.xe2x80x9d; Yokota, H., Sunwoo, J., Snikaya, M., van den Engh, G., Aebersold, R. (1999) xe2x80x9cSpin-Stretching of DNA and Protein Molecules for Detection by Fluorescence and Atomic Force Microscopy.xe2x80x9d
Also, the adjustment of the degree of immobilization through the chemical control of the pH-value was described for a large variety of different surfaces, see Allemand, J.-F., Bensimon, D., Julien, L., Bensimon, A, Croquette, V. (1997) Biophysical Journal, 73, 2064-2070 xe2x80x9cpH-Dependent Binding and Combing of DNA.xe2x80x9d
Yoshida, K., Yoshimoto, M., Sasaki, K., Ohnishi, T., Ushiki, T., Hitomik, J., Yamamoto, S., Sigeno, M. (1998) Biophysical Journal, 74, 1654-1657 xe2x80x9cFabrication of a New Substrate for Atomic Force Microscopic Observation of DNA Molecules from an Ultrasmooth Sapphire Plate.xe2x80x9d describes the hydrophilization of a sapphire surface treated with Na3PO4 aqueous solution. It is reported that the hydrophilic surface character after the wet treatment makes it easy for nucleic acid molecules to adhere to the substrate surface. Other approaches utilize the specific binding (chemisorption) of thiol-group terminated nucleic acid to gold surfaces and electrodes. Washizu, M., Kurosawa, O., Arai, I., Suzuki, S., Shimamoto, N. (1995) IEEE Trans. Industr. Appl., 31, 3, 447-456 xe2x80x9cApplications of Electrostatic Stretch-and-Positioning of DNA.xe2x80x9d reported on strong, covalent-like binding of nucleic acids to fresh aluminum electrodes in an alternating electrical field.
Oxygen plasma treatment is a well-known method to clean surfaces from organic impurities by oxidation, which supports the generation of OH-groups. U.S. Pat. No. 5,055,316 teaches the oxygen plasma supported tight binding of proteins to surfaces. Molecular tailoring of surfaces using a plasma treatment is disclosed in U.S. Pat. No. 5,876,753. A method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals was described in U.S. Pat. No. 5,369,012.
As mentioned above, various nucleic acid immobilization methods have been proposed. Most of them employ wet chemical treatment to modify the substrate. Therefore, the use of expensive chemical components is necessary, often not providing reproducible and permanent immobilization of nucleic acid molecules on that substrate.
Only a small variety of substrate materials can be employed using the above mentioned wet chemical treatment.
Accordingly, it is an object of the present invention to provide a process for immobilization of nucleic acid molecules on a substrate to overcome the drawbacks of prior art, especially to provide a process not requiring a wet chemical treatment of the substrate and to provide a reproducible process for the permanent immobilization of nucleic acid molecules on a substrate.
A further object underlying the present invention is to provide an immobilized nucleic acid which may be used in nucleic acid based nanotechnology.
The first object is solved by a process for immobilization of nucleic acid molecules on a substrate wherein the substrate is treated with atomic oxygen plasma prior to immobilizing the nucleic acid molecules thereon.
In a preferred embodiment the nucleic acid is selected from the group consisting of DNA,RNA, PNA (peptidic-NA), CNA (aminocyclohexylethane acid-NA), HNA (hexitol nucleic acids), p-RNA (pyranosyl-RNA), oligonucleotides, oligonucleotides of DNA, oligonucleotides of RNA, primers, A-DNA, B-DNA, Z-DNA, polynucleotides of DNA, polynucleotides of RNA, T-junctions of nucleic acids, domains of non-nucleic acid polymer-nucleic acid blockpolymers and combinations thereof. Suited non-nucleic acid polymers for blockcopolymers can be polypeptides, polysaccharides such as cellulose, or artificial polymers, such as polyethylene glycol, and are generally known to the person skilled in the art.
In another embodiment the nucleic acid is double-stranded or single-stranded.
In a further embodiment the nucleic acid is of natural character, modified, such as substituted with functional groups, non-modified or artificially generated.
In a still further embodiment the substrate is a single crystal surface or an amorphous surface.
More preferably the surface material is selected from the group comprising silicon oxides, glass, aluminum oxides, sapphire, perovskites, like SrTiO3, LaAlO3, NdGaO3, ZrO2 and derivatives thereof and doped and/or stabilized derivatives thereof, for example using Yttrium as stabilizer.
In a further preferred embodiment microwave generated oxygen plasma producing atomic oxygen or mixtures of gases containing oxygen are used. Preferred gases for admixture are all noble gases.
Alternatively high-voltage generated and/or UV-light emitting source generated oxygen plasma producing atomic oxygen or mixtures of gases containing oxygen are used.
Still a further embodiment is characterized in that the substrate is treated with atomic oxygen plasma for about 0.1 to 10 minutes.
It is preferred that the atomic oxygen plasma treatment is carried out using an oxygen pressure in the range of about 0.1 to 1.0 mbar, preferably 0.2 to 0.8 mbar.
The immobilization of the nucleic acid to the substrate can be adjusted by changing the intensity and duration of the plasma treatment. For example, using short time/low pressure conditions (pO2=0.4 mbar, t=4 mn) leads to a weak binding of DNA molecules to the surface, whilst using long-time/high-pressure conditions (pO2=0.8 mbar, t=8 mn) leads to a high density and strong binding of DNA molecules to the surface. These parameters correspond to a high-voltage power of 33 Watts at a frequency of 50 Hz. In the hands of the inventors these parameters lead to optimal results in terms of the cost to benefit-ratio. The pressures and times given here are meant as non-limiting examples of the invention. In fact, higher power levels can be used to reduce the minimum process time required to observe a significant binding effect. The individual protocols will vary depending on the machinery and the setup used for the immobilization process but can easily be determined by the person skilled in the art employing the general concept of the invention.
In a still further embodiment the nucleic acid to be immobilized on the substrate is present in an aqueous solution, for example a biological buffer solution.
Moreover it is preferred that the substrate is treated with the nucleic acid containing aqueous solution for at least a few seconds up to 5 minutes, preferably 1 to 2 minutes.
The second object is solved by an immobilized nucleic acid obtainable by a process according to the present invention.
In a still further aspect the object is solved by the use of the immobilized nucleic acid prepared according to the process of the present invention in nucleic acid based nanotechnology, such as nanoelectronics, like wires, biosensors, chips and the like.
Surprisingly, it was found that the process of the present invention is adjustable, highly reliable and fast, leads to long-term stable immobilization of nucleic acid molecules on various substrates, and is inexpensive. Moreover, the surface does not require any chemical treatment prior immobilization of nucleic acids thereon and therefore, the process of the present invention does not generate chemical waste. Additionally, it was surprisingly found that the process of the present invention is controllable in terms of the degree of immobilization of nucleic acid molecules. By changing the intensity and the duration of the plasma treatment the density of the nucleic acid molecules on the substrate surface and the density of binding sites is adjustable. The process of the present invention discloses a dry surface treatment to provide immobilization of nucleic acid molecules on a surface.
Also, it was surprisingly found that the immobilized nucleic acid obtainable by the process of the present invention can be used in nucleic acid based nanotechnology, such as nanoelectronics.
The nucleic acid herein referred to also as nucleic acid molecule may be either DNA or RNA. This interchangeability, which applies to many cases, resides in physiochemical similarities between DNA and RNA. Of course, any nucleic acids or derivatives thereof can also be used in the present invention such as, but not limited to, oligonucleotides of DNA and RNA, respectively, primers thereof and polynucleotides of each of said two nucleic acid species. Additionally, nucleic acids which can be used in the present invention may show various confirmations such as A-DNA, B-DNA and Z-DNA which differ mostly in the diameter and the particular kind of helix structure. Also domains of nucleic acids within larger units may be used. It is to be understood that any of the aforementioned nucleic acid species may be either in a double-stranded or single stranded form.